Isolated genomic polynucleotide fragments that encode human lipoprotein-associated phospholipase A2

ABSTRACT

The invention is directed to isolated genomic polynucleotide fragments that encode human lipoprotein-associated phospholipase A2, vectors and hosts containing the fragment and fragments hybridizing to noncoding regions as well as antisense oligonucleotides to these fragments. The invention is further directed to methods of using these fragments to obtain human lipoprotein-associated phospholipase A2 and to diagnose, treat, prevent and/or ameliorate a pathological disorder.

PRIORITY CLAIM

This application is a divisional application of application Ser. No.12/323,364, filed Nov. 25, 2008, which is a continuation application ofapplication Ser. No. 10/161,127, filed May 30, 2002, the contents ofwhich are incorporated herein by reference. application Ser. no.10/161,127 is claims priority under 35 USC 119(e) to provisionalapplication no. 60/294,404, filed May 30, 2001, the contents of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human human lipoprotein-associated phospholipase A2, vectorsand hosts containing these fragments and fragments hybridizing tononcoding regions as well as antisense oligonucleotides to thesefragments. The invention is further directed to methods of using thesefragments to obtain human lipoprotein-associated phospholipase A2 and todiagnose, treat, prevent and/or ameliorate a pathological and/or medicaldisorders.

BACKGROUND OF THE INVENTION

Chromosome 18 contains genes encoding, for example, GATA-bindingprotein-6, cadherin 2, retinoblastoma-binding protein 8, aquaporin-4,H+transporting ATP synthase and lipoprotein-associated phospholipase A2;the last of which will be discussed in more detail below.

Human Lipoprotein-Associated Phospholipase A2

Human lipoprotein-associated phospholipase A2, also known in the art asPlatelet Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase), isone of a family of enzymes that catalyze release of fatty acids frommembrane phospholipids and can thereby initiate synthesis ofproinflammatory mediators. During the conversion of LDL to its oxidisedform, lipoprotein-associated phospholipase A2 is responsible forhydrolysing the sn-2 ester of oxidatively modified phosphatidylcholineto give lyso-phosphatidylcholine and an oxidatively modified fatty acid.Both of these products of human lipoprotein-associated phospholipase A2action are potent chemoattractants for circulating monocytes. The enzymeappears to play a central role in the development of atherosclerosis andis regarded as an independent risk factor for coronary artery disease(Caslake et al., Atherosclerosis 150: 413-19, 2000). Specifically, thisenzyme is thought to be responsible for the accumulation of cells loadedwith cholesterol ester in the arteries, causing the characteristic‘fatty streak’ associated with the early stages of atherosclerosis.Recently, medicinal chemists have begun to design and preparelipoprotein-associated phospholipase A2 inhibitors for use in preventingor inhibiting progression of atherosclerotic diseases (See, for example,U.S. Pat. Nos. 5,981,252 and 5,968,818 Boyd et al., Bioorg. Med. Chem.10: 2557-61, 2000).

The level of PAF acetylhydrolase has been found to be altered in severaldisease states. For example, acquired deficiency of PAF acetylhydrolaseactivity has been reported in patients with systemic lupuserythematosus, stroke and asthma, and increased levels of PAF have beenreported in children with acute asthmatic attacks (see, for example,Hiramoto et al., Stroke 28: 2417-2420, 1997; Kruse et al., Am. J. Hum.Genet. 66: 1522-1530, 2000; Stafforini et al., J. Clin. Invest. 97:2784-2791, 1996). Miwa et al. (1988, J. Clin. Invest. 82:1983-1991) hasalso described an autosomal recessive form of PAF acetylhydrolasedeficiency which has been observed only in the Japanese population. PAFacetylhydrolase activity was absent in 4% of the Japanese population.This inherited deficiency is the result of a point mutation in exon 9and completely abolishes enzymatic activity. These patients suffer fromsevere asthma. Results from further studies indicated that the variantallele thr198 was found to be highly associated with total IgEconcentrations in an atopic population and with ‘asthma’ in an asthmaticpopulation (Kruse et al., 2000, Am. J. Hum. Genet. 66:1522-1530). Thevariant allele val379 was found to be highly associated with ‘specificsensitization’ in the atopic population and with ‘asthma’ in theasthmatic population.

The full length cDNA clone has been isolated (see U.S. Pat. Nos.5,981,252 and 5,968,818) and the DNA sequence has been determined Thecomplete amino acid sequence has been deduced from the DNA sequence. Itwas originally thought that the gene encoding the humanlipoprotein-associated phospholipase A2 polypeptide was located at genemap locus 6p21.2-p12.

OBJECTS OF THE INVENTION

Although cDNA encoding the above-disclosed protein, lipoproteinassociated phospholipase A2, has been isolated (e.g. see accession no.AX006795 and NM_(—)005084), its exact location on chromosome 18 andexon/intron/regulatory organization have not been determinedFurthermore, genomic DNA encoding the polypeptide has not been isolated.Noncoding sequences play a significant role in regulating the expressionof polypeptides as well as the processing of RNA encoding thesepolypeptides.

There is clearly a need for obtaining genomic polynucleotide sequencesencoding the lipoprotein-associated phospholipase A2 polypeptide.Therefore, it is an object of the invention to isolate such genomicpolynucleotide sequences.

There is also a need to develop means for identifying mutations,duplications, translocations, polysomies and mosaicism as may affect thelipoprotein associated phopholipase A2 gene.

SUMMARY OF THE INVENTION

The invention is directed to an isolated genomic polynucleotide, saidpolynucleotide obtainable from human chromosome 18 or chromosome 6having a nucleotide sequence at least 95% identical to a sequenceselected from the group consisting of:

-   -   (a) a polynucleotide encoding human lipoprotein-associated        phospholipase A2 depicted in SEQ ID NO:1.    -   (b) a polynucleotide consisting of SEQ ID NO:2 and 3, which        encodes human lipoprotein-associated phospholipase A2 depicted        in SEQ ID NO:1    -   (c) a polynucleotide which is a variant of SEQ ID NO:2 or 3;    -   (d) a polynucleotide which is an allelic variant of SEQ ID NO:2        or 3;    -   (e) a polynucleotide which encodes a variant of SEQ ID NO:1;    -   (f) a polynucleotide which hybridizes to any one of the        polynucleotides specified in (a)-(e) and    -   (g) a polynucleotide that is a reverse complement to the        polynucleotides specified in (a) to (f) and    -   (h) containing at least 10 transcription factor binding sites        selected from the group consisting

of AP1-C, AP1_Q4, AP4-Q5, DELTAEF1-01, GATA1_(—)04, GATA1-06, GATA2-02,GATA3_(—)02, GATA-C, LMO2COM-02, LYF1-01, MYOD_Q6, MZF_(—)01, NFAT_Q6,NKX25-01, S8-01, SOX5-01, TATA-C, and TCF11-01

as well as nucleic acid constructs, expression vectors and host cellscontaining these polynucleotide sequences.

The polynucleotides of the present invention may be used for themanufacture of a gene therapy for the prevention, treatment oramelioration of a medical condition such as asthma or systemic lupuserythematosus by adding an amount of a composition comprising saidpolynucleotide effective to prevent, treat or ameliorate said medicalcondition.

The invention is further directed to obtaining these polypeptides by

-   -   (a) culturing host cells comprising these sequences under        conditions that provide for the expression of said polypeptide        and    -   (b) recovering said expressed polypeptide.

The polypeptides obtained may be used to produce antibodies by

-   -   (a) optionally conjugating said polypeptide to a carrier        protein;    -   (b) immunizing a host animal with said polypeptide or        peptide-carrier protein conjugate of step (b) with an adjuvant        and    -   (c) obtaining antibody from said immunized host animal.

The invention is further directed to polynucleotides that hybridize tononcoding regions of said polynucleotide sequences as well as antisenseoligonucleotides to these polynucleotides as well as antisense mimetics.The antisense oligonucleotides or mimetics may be used for themanufacture of a medicament for prevention, treatment or amelioration ofa medical condition, such as atherosclerosis. The invention is furtherdirected to kits comprising these polynucleotides and kits comprisingthese antisense oligonucleotides or mimetics.

In a specific embodiment, the noncoding regions are transcriptionregulatory regions. The transcription regulatory regions may be used toproduce a heterologous peptide by expressing in a host cell, saidtranscription regulatory region operably linked to a polynucleotideencoding the heterologous polypeptide and recovering the expressedheterologous polypeptide.

The polynucleotides of the present invention may be used to diagnose apathological condition such as asthma in a subject comprising

-   -   (a) determining the presence or absence of a mutation in the        polynucleotides of the present invention and    -   (b) diagnosing a pathological condition or a susceptibility to a        pathological condition based on the presence or absence of said        mutation.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to isolated genomic polynucleotide fragmentsthat encode human lipoprotein-associated phospholipase A2, which in aspecific embodiment is the human lipoprotein-associated phospholipase A2gene, as well as vectors and hosts containing these fragments andpolynucleotide fragments hybridizing to noncoding regions, as well asantisense oligonucleotides to these fragments.

As defined herein, a “gene” is the segment of DNA involved in producinga polypeptide chain; it includes regions preceding and following thecoding region, as well as intervening sequences (introns) betweenindividual coding segments (exons).

As defined herein “isolated” refers to material removed from itsoriginal environment and is thus altered “by the hand of man” from itsnatural state. An isolated polynucleotide can be part of a vector, acomposition of matter or can be contained within a cell as long as thecell is not the original environment of the polynucleotide.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes genomic DNA and synthetic DNA.The DNA may be double-stranded or single-stranded and if single strandedmay be the coding strand or non-coding strand.

The portion of the human lipoprotein-associated phospholipase A2 genethat contains exons 3 to 11 is 24696 base pairs in length (SEQ ID NO:2)(see Table 1 below for location of exons). Exons 1 and 2 are encoded inSEQ ID NO:3 which is 17889 base pairs in length. The gene is situated inchromosome 18 genomic clone AC008104; contigs 21 (SEQ ID NO:2) and 20(SEQ ID NO: 3) of gi 8072415.

The polynucleotides of the invention have at least a 95% identity andmay have a 96%, 97%, 98% or 99% identity to the polynucleotides depictedin SEQ ID NO:2 & 3 as well as the polynucleotides in reverse senseorientation, or the polynucleotide sequences encoding the humanlipoprotein-associated phospholipase A2 polypeptide depicted in SEQ IDNO:1.

A polynucleotide having 95% “identity” to a reference nucleotidesequence of the present invention, is identical to the referencesequence except that the polynucleotide sequence may include, onaverage, up to five point mutations per each 100 nucleotides of thereference nucleotide sequence encoding the polypeptide. In other words,to obtain a polynucleotide having a nucleotide sequence at least 95%identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence, the ORF (openreading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.(1990) 6:237-245). In a sequence alignment the query and subjectsequences are both DNA sequences. An RNA sequence can be compared byconverting U's to T's. The result of said global sequence alignment isin percent identity. Preferred parameters used in a FASTDB alignment ofDNA sequences to calculate percent identity are: Matrix=Unitary,k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject nucleotide sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identify,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequenceare calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total numbers of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are made forpurposes of the present invention.

A polypeptide that has an amino acid sequence at least, for example, 95%“identical” to a query amino acid sequence is identical to the querysequence except that the subject polypeptide sequence may include onaverage, up to five amino acid alterations per each 100 amino acids ofthe query amino acid sequence. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a query aminoacid sequence, up to 5% of the amino acid residues in the subjectsequence may be inserted, deleted, (indels) or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the referenced sequence or in one or morecontiguous groups within the reference sequence.

A preferred method for determining the best overall match between aquery sequence (a sequence of the present invention) and a subjectsequence, also referred to as a global sequence alignment, can bedetermined using the FASTDB computer program based on the algorithm ofBrutlag et al. (Com. App. Biosci. (1990) 6:237-245). In a sequencealignment, the query and subject sequence are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0, CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

The invention also encompasses polynucleotides that hybridize to thepolynucleotides depicted in SEQ ID NO: 2 or 3. A polynucleotide“hybridizes” to another polynucleotide, when a single-stranded form ofthe polynucleotide can anneal to the other polynucleotide under

the appropriate conditions of temperature and solution ionic strength(see Sambrook et al., supra). The conditions of temperature and ionicstrength determine the “stringency” of the hybridization. Forpreliminary screening for homologous nucleic acids, low stringencyhybridization conditions,corresponding to a temperature of 42 C, can be used, e.g., 5X SSC, 0.1%SDS, 0.25% milk, and no formamide; or 40% formamide, 5X SSC, 0.5%SDS). Moderate stringency hybridization conditions correspond to ahigher temperature of 55 C, e.g., 40% formamide, with 5X or 6X SCC. Highstringency hybridization conditions correspond to the highesttemperature of 65 C, e.g., 50% formamide, 5X or 6X SCC. Hybridizationrequires that the two nucleic acids contain complementary sequences,although depending on the stringency of the hybridization, mismatchesbetween bases are possible. The appropriate stringency for hybridizingnucleic acids depends on the length of the nucleic acids and the degreeof complementation, variables well known in the art. The greater thedegree of similarity or homology between two nucleotide sequences, thegreater the value of T_(m) for hybrids of nucleic acids having thosesequences. The relative stability (corresponding to higher T_(m)) ofnucleic acid hybridizations decreases in the following order: RNA:RNA,DNA:RNA, DNA:DNA.

Polynucleotide and Polypeptide Variants

The invention is directed to both polynucleotide and polypeptidevariants. A “variant” refers to a polynucleotide or polypeptidediffering from the polynucleotide or polypeptide of the presentinvention, but retaining essential properties thereof. Generally,variants are overall closely similar and in many regions, identical tothe polynucleotide or polypeptide of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred.

The invention also encompasses allelic variants of said polynucleotides.An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequences depicted in SEQ ID NO:1 by an insertion or deletionof one or more amino acid residues and/or the substitution of one ormore amino acid residues by different amino acid residues. Preferably,amino acid changes are of a minor nature, that is conservative aminoacid substitutions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of one to about 30amino acids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine) Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, as well as these in reverse.

Noncoding Regions

The invention is further directed to polynucleotide fragments containingor hybridizing to noncoding regions of the human lipoprotein-associatedphospholipase A2 gene. These include but are not limited to an intron, a5′-non-coding region, a 3′-non-coding region and splice junctions (seeTable 1), as well as transcription factor binding sites (see Table 2).The polynucleotide fragments may be a short polynucleotide fragmentwhich is between about 8 nucleotides to about 40 nucleotides in length.Such shorter fragments may be useful for diagnostic purposes. Such shortpolynucleotide fragments are also preferred with respect topolynucleotides containing or hybridizing to polynucleotides containingsplice junctions. Alternatively larger fragments, e.g., of about 50,150, 500, 600 or about 2000 nucleotides in length may be used.

TABLE 1 Exon/Intron Regions of the Liporotein Related Phospholipase A2Gene in Contigs 20 (17889 base pairs) and 21 (24696 base pairs) ofAC008104, gi 8072415 (reverse strand coding). Exon Nucleotide no.Peptide Amino Acid no. Contig (stop codon) 12311-12313 21 11 12314-12448 441-397 21 10  12905-13054 396-347 21 9 15744-15914 346-29021 8 17080-17169 289-260 21 7 18296-18409 259-222 21 6 19247-19372221-180 21 5 20022-20090 179-157 21 4 22213-22305 156-126 21 324136-24279 125-78  21 2  4-129 77-36 20 1 5819-5923 35-1  20

TABLE 2 Transcription Factor Binding Sites on the Lipoprotein RealatedPhospholipase A2 Gene. Transcription Factor No. of Binding Sites AP1_C14 AP1_Q4 4 AP4_Q5 4 DELTAEF1_01 5 GATA1_04 7 GATA1_06 8 GATA2_02 5GATA3_02 4 GATA_C 7 LMO2COM_02 4 LYF1_01 9 MYOD_Q6 9 MZF1_01 14 NFAT_Q610 NKX25_01 16 S8_01 7 SOX5_01 2 TATA_C 7 TCF11_01 46

In a specific embodiment, such noncoding sequences are expressioncontrol sequences. These include but are not limited to DNA regulatorysequences, such as promoters, enhancers, repressors, terminators, andthe like, that provide for the regulation of expression of a codingsequence in a host cell. In eukaryotic cells, polyadenylation signalsare also control sequences.

In a more specific embodiment of the invention, the expression controlsequences may be operatively linked to a polynucleotide encoding aheterologous polypeptide. Such expression control sequences may be about50-200 nucleotides in length and specifically about 50, 100, 200, 500,600, 1000 or 2000 nucleotides in length. A transcriptional controlsequence is “operatively linked” to a polynucleotide encoding aheterologous polypeptide sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence. The term “operatively linked” includes havingan appropriate start signal (e.g., ATG) in front of the polynucleotidesequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence and production of the desired productencoded by the polynucleotide sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted upstream (5′) of andin reading frame with the gene.

The invention is further directed to antisense oligonucleotides andmimetics to these polynucleotide sequences. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes the mature polypeptides of thepresent invention, is used to design an antisense RNA oligonucleotide offrom about 10 to 40 base pairs in length. A DNA oligonucleotide isdesigned to be complementary to a region of the gene involved intranscription or RNA processing (triple helix (see Lee et al., Nucl.Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); andDervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of said polypeptides.

Expression of Polypeptides

Isolated Polynucleotide Sequences

The human chromosome 18 genomic clone of accession number AC008104 hasbeen discovered to contain the human lipoprotein-associatedphospholipase A2 gene by Genscan analysis (Burge et al., 1997, J. Mol.Biol. 268:78-94), BLAST2 and TBLASTN analysis (Altschul et al., 1997,Nucl. Acids Res. 25:3389-3402), in which the sequences of AC008104 werecompared to the human lipoprotein-associated phospholipase A2 cDNAsequence, accession number NM_(—)005084 or AX006795.

The cloning of the nucleic acid sequences of the present invention fromsuch genomic DNA can be effected, e.g., by using the well knownpolymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligated activatedtranscription (LAT) and nucleic acid sequence-based amplification(NASBA) or long range PCR may be used. In a specific embodiment, 5′- or3′-non-coding portions of the gene may be identified by methodsincluding but are not limited to, filter probing, clone enrichment usingspecific probes and protocols similar or identical to 5′- and 3′-“RACE”protocols which are well known in the art. For instance, a methodsimilar to 5′-RACE is available for generating the missing 5′end of adesired full-length transcript. (Fromont-Racine et al., 1993, Nucl.Acids Res. 21:1683-1684).

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired human lipoprotein-associatedphospholipase A2 gene may be accomplished in a number of ways. Forexample, if an amount of a portion of a human lipoprotein-associatedphospholipase A2 gene or its specific RNA, or a fragment thereof, isavailable and can be purified and labeled, the generated DNA fragmentsmay be screened by nucleic acid hybridization to the labeled probe(Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975,Proc. Natl. Acad. Sci. U.S.A. 72:3961). The present invention providessuch nucleic acid probes, which can be conveniently prepared from thespecific sequences disclosed herein, e.g., a hybridizable probe having anucleotide sequence corresponding to at least a 10, and preferably a 15,nucleotide fragment of the sequences depicted in SEQ ID NO:2 or b.Preferably, a fragment is selected that is highly unique to thepolypeptides of the invention. Those DNA fragments with substantialhomology to the probe will hybridize. As noted above, the greater thedegree of homology, the more stringent hybridization conditions can beused. In one embodiment, low stringency hybridization conditions areused to identify a homologous human lipoprotein-associated phospholipaseA2 polynucleotide. However, in a preferred aspect, and as demonstratedexperimentally herein, a nucleic acid encoding a polypeptide of theinvention will hybridize to a nucleic acid derived from thepolynucleotide sequence depicted in SEQ ID NO:2 or b or a hybridizablefragment thereof, under moderately stringent conditions; morepreferably, it will hybridize under high stringency conditions.

Alternatively, the presence of the gene may be detected by assays basedon the physical, chemical, or immunological properties of its expressedproduct. For example, cDNA clones, or DNA clones which hybrid-select theproper mRNAs, can be selected which produce a protein that, e.g., hassimilar or identical electrophoretic migration, isoelectric focusingbehavior, proteolytic digestion maps, or antigenic properties as knownfor the human lipoprotein-associated phospholipase A2 polynucleotide.

A gene encoding human lipoprotein-associated phospholipase A2polypeptide can also be identified by mRNA selection, i.e., by nucleicacid hybridization followed by in vitro translation. In this procedure,fragments are used to isolate complementary mRNAs by hybridizationImmunoprecipitation analysis or functional assays of the in vitrotranslation products of the products of the isolated mRNAs identifiesthe mRNA and, therefore, the complementary DNA fragments, that containthe desired sequences.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide sequence containing the exon/intron segments of thehuman lipoprotein-associated phospholipase A2 gene operably linked toone or more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

The invention is further directed to a nucleic acid construct comprisingexpression control sequences derived from SEQ ID NO: 2 or 3 and aheterologous polynucleotide sequence.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term expression cassette when the nucleic acid constructcontains all the control sequences required for expression of a codingsequence of the present invention. The term “coding sequence” is definedherein as a portion of a nucleic acid sequence which directly specifiesthe amino acid sequence of its protein product. The boundaries of thecoding sequence are generally determined by a ribosome binding site(prokaryotes) or by the ATG start codon (eukaryotes) located justupstream of the open reading frame at the 5′-end of the mRNA and atranscription terminator sequence located just downstream of the openreading frame at the 3′-end of the mRNA. A coding sequence can include,but is not limited to, DNA, cDNA, and recombinant nucleic acidsequences.

The isolated polynucleotide of the present invention may be manipulatedin a variety of ways to provide for expression of the polypeptide.Manipulation of the nucleic acid sequence prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizingrecombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which regulate the expression of the polynucleotide.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Komaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80: 21-25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulansacetamidase, Fusarium oxysporum trypsin-like protease (WO 96/00787),NA2-tpi (a hybrid of the promoters from the genes encoding Aspergillusniger neutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the3′-terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase,Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes encoding Aspergillus oryzae TAKA amylase and Aspergillusnidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from theSaccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomycescerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiaealpha-factor, and the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region, whichcodes for an amino acid sequence linked to the amino terminus of thepolypeptide which can direct the encoded polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not normallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to obtain enhanced secretion of thepolypeptide. However, any signal peptide coding region which directs theexpressed polypeptide into the secretory pathway of a host cell ofchoice may be used in the present invention.

An effective signal peptide coding region for bacterial host cells isthe signal peptide coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), or theBacillus subtilis prsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57: 109-137.

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from the Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene,Aspergillus niger glucoamylase gene, Rhizomucor miehei asparticproteinase gene, Humicola lanuginosa cellulase gene, or Humicolalanuginosa lipase gene.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from theBacillus subtilis alkaline protease gene (aprE), the Bacillus subtilisneutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factorgene, the Rhizomucor miehei aspartic proteinase gene, or theMyceliophthora thermophila laccase gene (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the polynucleotide of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5Õ-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents from other species.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

For integration into the host cell genome, the vector may rely on thepolynucleotide sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional polynucleotide sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB 110, pE194, pTA1060, andpAM§1 permitting replication in Bacillus. Examples of origins ofreplication for use in a yeast host cell are the 2 micron origin ofreplication, ARS1, ARS4, the combination of ARS1 and CEN3, and thecombination of ARS4 and CEN6. The origin of replication may be onehaving a mutation which makes its functioning temperature-sensitive inthe host cell (see, e.g., Ehrlich, 1978, Proceedings of the NationalAcademy of Sciences USA 75: 1433).

More than one copy of a polynucleotide sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the polynucleotide sequencecan be obtained by integrating at least one additional copy of thesequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Useful unicellularcells are bacterial cells such as gram positive bacteria including, butnot limited to, a Bacillus cell, e.g., Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces cell,e.g., Streptomyces lividans or Streptomyces murinus, or gram negativebacteria such as E. coli and Pseudomonas sp. In a preferred embodiment,the bacterial host cell is a Bacillus lentus, Bacillus licheniformis,Bacillus stearothermophilus or Bacillus subtilis cell. In anotherpreferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian cell (e.g., humancell), an insect cell, a plant cell or a fungal cell. Mammalian hostcells that could be used include but are not limited to human Hela, 293,H9 and Jurkat cells, mouse NIH3t3 and C127 cells, Cos 1, Cos 7 and CV1,quail QC1-3 cells, mouse L cells and Chinese Hamster ovary (CHO) cells.These cells may be transfected with a vector containing atranscriptional regulatory sequence, a protein coding sequence andtranscriptional termination sequences. Alternatively, the polypeptidecan be expressed in stable cell lines containing the polynucleotideintegrated into a chromosome. The co-transfection with a selectablemarker such as dhfr, gpt, neomycin, hygromycin allows the identificationand isolation of the transfected cells.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra). The fungal host cell may also be a yeast cell. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980). The fungal host cell may also be a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. In a specific embodiment, an enzyme assay may beused to determine the activity of the polypeptide. For example, humanlipoprotein-associated phospholipase A2 can be assayed by its ability torelease fatty acids from phospholipids. Caslake et al (Atherosclerosis150: 413-19, 2000) have described a specific immunoassay for the enzyme.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Antibodies

According to the invention, the human lipoprotein-associatedphospholipase A2 polypeptide produced according to the method of thepresent invention may be used as an immunogen to generate any of theseantibodies. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

Various procedures known in the art may be used for the production ofantibodies. For the production of antibody, various host animals can beimmunized by injection with the polypeptide thereof, including but notlimited to rabbits, mice, rats, sheep, goats, etc. In one embodiment,the polypeptide or fragment thereof can optionally be conjugated to animmunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the humanlipoprotein-associated phospholipase A2 polypeptide, any technique thatprovides for the production of antibody molecules by continuous celllines in culture may be used. These include but are not limited to thehybridoma technique originally developed by Kohler and Milstein (1975,Nature 256:495-497), as well as the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In an additional embodiment of the invention,monoclonal antibodies can be produced in germ-free animals utilizingrecent technology (PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, J.Bacteriol. 159-870; Neuberger et al., 1984, Nature 312:604-608; Takedaet al., 1985, Nature 314:452-454) by splicing the genes from a mouseantibody molecule specific for the human lipoprotein-associatedphospholipase A2 polypeptide together with genes from a human antibodymolecule of appropriate biological activity can be used; such antibodiesare within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce polypeptide-specific single chain antibodies. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for the humanlipoprotein-associated phospholipase A2 polypeptide.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab'fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂, fragment,

and the Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,

immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention. For example, to select antibodies which recognize aspecific epitope of a particular polypeptide, one may assay generatedhybridomas for a product which binds to a particular polypeptidefragment containing such epitope. For selection of an antibody specificto a particular polypeptide from a particular species of animal, one canselect on the basis of positive binding with the polypeptide expressedby or isolated from cells of that species of animal.

Immortal, antibody-producing cell lines can also be created bytechniques other than fusion, such as direct transformation of Blymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus.See, e.g., M. Schreier et al., “Hybridoma Techniques” (1980); Hammerlinget al.,

“Monoclonal Antibodies And T-cell Hybridomas” (1981); Kennett etal.,“Monoclonal Antibodies” (1980); see also U.S. Pat. Nos. 4,341,761;4,399,121; 4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500;4,491,632; 4,493,890.

Uses of Polynucleotides

Diagnostics

Polynucleotides containing noncoding regions of SEQ ID NO:2 or 3 may beused as probes for detecting mutations from samples from a patient.Genomic DNA may be isolated from the patient. A mutation(s) may bedetected by Southern blot analysis, specifically by hybridizingrestriction digested genomic DNA to various probes and subjecting toagarose electrophoresis. Alternatively, these polynucleotides may beused as PCR primers and be used to amplify the genomic DNA isolated fromthe patients. Additionally, primers may be obtained by routine or longrange PCR that yield products containing contiguous intron/exon sequenceand products containing more than one exon with intervening intron. Thesequence of the amplified genomic DNA from the patient may be determinedusing methods known in the art. Such probes may be between 10-100nucleotides in length and may preferably be between 20-50 nucleotides inlength.

Thus the invention is thus directed to kits comprising thesepolynucleotide probes. In a specific embodiment, these probes arelabeled with a detectable substance.

Antisense Oligonucleotides and Mimetics

The antisense oligonucleotides or mimetics of the present invention maybe used to decrease levels of a polypeptide. For example, humanlipoprotein-associated phospholipase A2 has been found to be associatedwith atherosclerosis and diabetes. Therefore, the humanlipoprotein-associated phospholipase A2 antisense oligonucleotides ofthe present invention could be used to inhibit progression ofatherosclerosis, including coronary artery disease.

The antisense oligonucleotides of the present invention may beformulated into pharmaceutical compositions. These compositions may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal

delivery), pulmonary, e.g., by inhalation or insufflation of powders oraerosols, including by nebulizer; intratracheal, intranasal, epidermaland transdermal), oral or parenteral. Parenteral administration includesintravenous, intraarterial, subcutaneous, intraperitoneal orintramuscularinjection or infusion; or intracranial, e.g., intrathecal orintraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers ,dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the active

ingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention, the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days to

several months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀ as found to be effective in invitro and in vivo animal models.

In general, dosage is from 0.01 ug to 10 g per kg of body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand

concentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligonucleotide is administered in maintenance doses,ranging from 0.01 ug to 10 g per kg of body weight, once or more daily,to once every 20 years.

Gene Therapy

Human lipoprotein-associated phospholipase A2 deficiency states havebeen described (Yamada et al., Metabolism 47: 177-81, 1998). Therefore,the human lipoprotein-associated phospholipase A2 gene may be used tovia gene therapy to correct any such deficiency state or disordersassociated with such deficiency states (e.g., asthma).

As described herein, the polynucleotide of the present invention may beintroduced into a patient's cells for therapeutic uses. As will bediscussed in further detail below, cells can be transfected using anyappropriate means, including viral vectors, as shown by the example,chemical transfectants, or physico-mechanical methods such aselectroporation and direct diffusion of DNA. See, for example, Wolff,Jon A, et al., “Direct gene transfer into mouse muscle in vivo,”Science, 247, 1465-1468, 1990; and Wolff, Jon A, “Human dystrophinexpression in mdx mice after intramuscular injection of DNA constructs,”Nature, 352, 815-818, 1991. As used herein, vectors are agents thattransport the gene into the cell without degradation and include apromoter yielding expression of the gene in the cells into which it isdelivered. As will be discussed in further detail below, promoters canbe general promoters, yielding expression in a variety of mammaliancells, or cell specific, or even nuclear versus cytoplasmic specific.These are known to those skilled in the art and can be constructed usingstandard molecular biology protocols. Vectors have been divided into twoclasses:

a) Biological agents derived from viral, bacterial or other sources.

b) Chemical physical methods that increase the potential for geneuptake, directly introduce the gene into the nucleus or target the geneto a cell receptor.

Biological Vectors

Viral vectors have higher transaction (ability to introduce genes)abilities than do most chemical or physical methods to introduce genesinto cells. Vectors that may be used in the present invention includeviruses, such as adenoviruses, adeno associated virus (AAV), vaccinia,herpesviruses, baculoviruses and retroviruses, bacteriophages, cosmids,plasmids, fungal vectors and other recombination vehicles typically usedin the art which have been described for expression in a variety ofeukaryotic and prokaryotic hosts, and may be used for gene therapy aswell as for simple protein expression. Polynucleotides are inserted intovector genomes using methods well known in the art.

Retroviral vectors are the vectors most commonly used in clinicaltrials, since they carry a larger genetic payload than other viralvectors. However, they are not useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation. Pox viralvectors are large and have several sites for inserting genes, they arethermostable and can be stored at room temperature.

Examples of promoters are SP6, T4, T7, SV40 early promoter,cytomegalovirus (CMV) promoter, mouse mammary tumor virus (MMTV)steroid-inducible promoter, Moloney murine leukemia virus (MMLV)promoter, phosphoglycerate kinase (PGK) promoter, and the like.Alternatively, the promoter may be an endogenous adenovirus promoter,for example the E1 a promoter or the Ad2 major late promoter (MLP).Similarly, those of ordinary skill in the art can construct adenoviralvectors utilizing endogenous or heterologous poly A addition signals.

Plasmids are not integrated into the genome and the vast majority ofthem are present only from a few weeks to several months, so they aretypically very safe. However, they have lower expression levels thanretroviruses and since cells have the ability to identify and eventuallyshut down foreign gene expression, the continuous release of DNA fromthe polymer to the target cells substantially increases the duration offunctional expression while maintaining the benefit of the safetyassociated with non-viral transfections.

Chemical/Physical Vectors

Other methods to directly introduce genes into cells or exploitreceptors on the surface of cells include the use of liposomes andlipids, ligands for specific cell surface receptors, cell receptors, andcalcium phosphate and other chemical mediators, microinjections directlyto single cells, electroporation and homologous recombination. Liposomesare commercially available from Gibco BRL, for example, asLIPOFECTIN^(..) and LIPOFECTACE^(..), which are formed of cationiclipids such as N-[1-(2,3 dioleyloxy)-propyl]-n,n,n-trimethylammoniumchloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).Numerous methods are also published for making liposomes, known to thoseskilled in the art. For example, Nucleic acid-Lipid Complexes—Lipidcarriers can be associated with naked nucleic acids (e.g., plasmid DNA)to facilitate passage through cellular membranes. Cationic, anionic, orneutral lipids can be used for this purpose. However, cationic lipidsare preferred because they have been shown to associate better with DNAwhich, generally, has a negative charge. Cationic lipids have also beenshown to mediate intracellular delivery of plasmid DNA (Felgner andRingold, Nature 337:387 (1989)). Intravenous injection of cationiclipid-plasmid complexes into mice has been shown to result in expressionof the DNA in lung (Brigham et al., Am. J. Med. Sci.298:278 (1989)). Seealso, Osaka et al., J. Pharm. Sci. 85(6):612-618 (1996); San et al.,Human Gene Therapy 4:781-788 (1993); Senior et al., Biochemica etBiophysica Acta 1070:173-179 (1991); Kabanov and Kabanov, BioconjugateChem. 6:7-20 (1995); Remy et al., Bioconjugate Chem. 5:647-654 (1994);Behr, J-P., Bioconjugate Chem 5:382-389 (1994); Behr et al., Proc. Natl.Acad. Sci., USA 86:6982-6986 (1989); and Wyman et al., Biochem.36:3008-3017 (1997).

Cationic lipids are known to those of ordinary skill in the art.Representative cationic lipids include those disclosed, for example, inU.S. Pat. No. 5,283,185; and e.g., U.S. Pat. No. 5,767,099. In apreferred embodiment, the cationic lipid is N.sup.4 -sperminecholesteryl carbamate (GL-67) disclosed in U.S. Pat. No. 5,767,099.Additional preferred lipids include N4 Dspermidine cholestryl carbamate(GL-53) and 1-(N4 -spermind) -2,3-dilaurylglycerol carbamate (GL-89).

The vectors of the invention may be targeted to specific cells bylinking a targeting molecule to the vector. A targeting molecule is anyagent that is specific for a cell or tissue type of interest, includingfor example, a ligand, antibody, sugar, receptor, or other bindingmolecule.

Invention vectors may be delivered to the target cells in a suitablecomposition, either alone, or complexed, as provided above, comprisingthe vector and a suitably acceptable carrier. The vector may bedelivered to target cells by methods known in the art, for example,intravenous, intramuscular, intranasal, subcutaneous, intubation,lavage, and the like. The vectors may be delivered via in vivo or exvivo applications. In vivo applications involve the directadministration of an adenoviral vector of the invention formulated intoa composition to the cells of an individual. Ex vivo applicationsinvolve the transfer of the adenoviral vector directly to harvestedautologous cells which are maintained in vitro, followed byreadministration of the transduced cells to a recipient.

In a specific embodiment, the vector is transfected intoantigen-presenting cells. Suitable sources of antigen-presenting cells(APCs) include, but are not limited to, whole cells such as dendriticcells or macrophages; purified MHC class I molecule complexed tobeta2-microglobulin and foster antigen-presenting cells. In a specificembodiment, the vectors of the present invention may be introduced intoT cells or B cells using methods known in the art (see, for example,Tsokos and Nepom, 2000, J. Clin. Invest. 106:181-183).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entireties.

1. An isolated polynucleotide probe or primer, wherein said probe orprimer is a fragment of a polynucleotide which is at least 99% identicalto SEQ ID NO:2 and wherein said polynucleotide probe or primer comprisesat least 20 contiguous nucleotides of SEQ ID NO:2 or its full complementwhich contains a transcription factor binding site at an AP1_C site atnucleotides 3731-3741, 4338-4348, 5013-5025, 15556-15566, 15596-15606,19533-19543, 35142-35152, 38600-38610, 40256-40266, 41192-41202 or41585-41595 of SEQ ID NO:2; an AP4_Q5 site at nucleotides 32929-32945or38920-38936 of SEQ ID NO:2; a GATA1_(—)04 site at nucleotides25798-25810, 27827-27839, 28501-28513, 31629-31641, 32034-32046,32169-32181, 33274-33286, 33538-33550or 41958-41970 of SEQ ID NO:2; aGATA1_(—)06 site at nucleotides 30717-30729, 31464-31476, 35301-35313 or41365-41377 of SEQ ID NO:2; a GATA2_(—)02 site at nucleotides 3486-3498,3609-3621, 9107-9119, 10637-10659, 18349-18361, 20858-20870,32236-32248, 34609-34621, 35665-35677, 36781-36793 or 42161-42173 of SEQID NO:2; a GATA3_(—)02 site at nucleotides 24794-24806, 25834-25846,31071-31083, 31973-31985 or 35551-35563 of SEQ ID NO:2; a GATA_C site atnucleotides 343-355, 650-662;3370-3382, 4507-4519, 5160-5172, 7500-7512,8422-8434, 10595-10607, 17905-17917, 22600-22612, 24775-24797,27763-27775, 30027-30039, 30541-30553, 33695-33707, 33935-33947 or41929-41941 of SEQ ID NO:2; a MZF1_(—)01 site at nucleotides 2907-2915,4766-4774;10854-10862, 11216-11224, 12675-12683, 15186-15194,27471-27479, 28548-28556 or 33107-33115 of SEQ ID NO:2; an NFAT_Q6 siteat nucleotides 1557-1575, 3717-3735, 3779-3797, 4621-4639, 5588-5606,7380-7398, 9288-9306, 11523-11541, 12221-12239, 13103-13121,13236-13254, 14475-14793, 16579-16597, 19812-19830, 21660-21678,26158-26176, 27106-27124, 27872-27890, 28104-28122, 29107-29125,32393-32411, 32816-32834, 33285-33303, 34446-35564 or 40661-40679 of SEQID NO:2; a NKX25_(—)01 site at nucleotides 4402-4416, 8109-8123 or33218-33232; a 58_(—)01 site at nucleotides 2836-2848, 3390-3402,3393-3405, 3872-3884, 3875-3887, 4227-4239, 12123-12137, 13569-13583,15144-151458, 17041-17055, 21032-21046 or 28959-28973 of SEQ ID NO:2; aS8_(—)01 site at nucleotides 12775-12787, 13388-13400, 14000-14012,18248-18260, 25893-25905, 25896-25908, 25948-25960, 27231-27243 or27497-27509 of SEQ ID NO:2; a SOX5_(—)01 site at nucleotides 42438-42454of SEQ ID NO:2; a TATA_C site at nucleotides 25236-25242, 25245-25261,or 41494-41510 of SEQ ID NO:2 or a TCF11_(—)01 site at nucleotides2093-2099, 10078-10084, 10792-10798, 14200-14206, 15162-15168,28408-28414, 32852-32858, 34893-34899, 38599-38905 or 41773-41779 of SEQID NO:2.
 2. A method for isolating the polynucleotide of claim 1comprising (a) isolating genomic DNA from a sample; (b) providingprimers, probes and optionally polymerase and (c) incubating (a) and (b)under conditions promoting the isolation of said nucleic acid molecule.3. A kit comprising the polynucleotide of claim
 1. 4. The polynucleotideaccording to claim 1, wherein said polynucleotide is an RNA or DNA.